Sabra et al.

BMC Neurology (2016) 16:254

DOI 10.1186/s12883-016-0778-x

RESEARCH ARTICLE Open Access

Assessment of platelet function in patients

with stroke using multiple electrode
platelet aggregometry: a prospective
observational study
Ahmed Sabra1,2,3, Sophia N. Stanford1,2, Sharon Storton2, Matthew Lawrence1,2, Lindsay DSilva1,2,
Roger H. K. Morris4, Vanessa Evans2, Mushtaq Wani5, John F. Potter6 and Phillip A. Evans1,2,7*

Abstract
Background: There is a link between high on-treatment platelet reactivity (HPR) and adverse vascular events in
stroke. This study aimed to compare multiple electrode platelet aggregometry (MEA), in healthy subjects and
ischaemic stroke patients, and between patients naive to antiplatelet drugs (AP) and those on regular low dose AP.
We also aimed to determine prevalence of HPR at baseline and at 35 days after loading doses of aspirin.
Methods: Patients with first ever ischaemic stroke were age and sex-matched to a healthy control group. Three
venous blood samples were collected: on admission before any treatment given (baseline); at 24 h and 35 days
after standard treatment. MEA was determined using a Mutliplate analyser and agonists tested were arachidonic
acid (ASPI), adenosine diphosphate (ADP) and collagen (COL).
Results: Seventy patients (mean age 73 years [SD 13]; 42 men, 28 women) were age and sex-matched to 72
healthy subjects. Thirty-three patients were on antiplatelet drugs (AP) prior to stroke onset and 37 were AP-naive.
MEA results for all agonists were significantly increased in AP-naive patients compared to healthy subjects: ADP
98 31 vs 81 24, p < 0.005; ASPI 117 31 vs 98 27, p < 0.005; COL 100 25 vs 82 20, p < 0.005. For patients on
long term AP, 33% (10/30) of patients were considered aspirin-resistant. At 35 days following loading doses of
aspirin, only 11.1% were aspirin resistant based on an ASPI cut-off value of 40 AU*min.
Conclusions: Many patients receiving low dose aspirin met the criteria of aspirin resistance but this was much
lower at 35 days following loading doses of aspirin. Future studies are needed to establish the causes of HPR and
potential benefits of individualizing AP treatment based on platelet function testing.
Keywords: Ischaemic stroke, Multiple electrode platelet aggregometry, Platelet function, Antiplatelet therapy,
Aspirin, Clopidogrel, Aspirin resistance

Background drugs, which are the mainstream therapy in the primary

Platelets play a major role in arterial thrombus formation
and therefore in the pathophysiology of ischaemic stroke
[14]. Excessive platelet activation leads to increased
thrombin generation and potentially abnormal thrombus
formation [5]. Hence the importance of oral antiplatelet
* Correspondence: phillip.evans2@wales.nhs.uk
and secondary prevention of cerebrovascular disease
[6, 7]. Currently, the antiplatelet (AP) drugs most
widely used are aspirin and clopidogrel, and multiple
electrode platelet aggregometry (MEA) has been used
to study their effects on platelet function [811].
MEA has also been used to investigate the effects of
non-opioid analgesics [12], anticoagulants [13], antifi-

Equal contributors
1
Medical School, Swansea University, Swansea, UK brinolytics [14] and temperature [15] on platelet
2
NISCHR Haemostasis Biomedical Research Unit, Morriston Hospital, Swansea, aggregation.
UK
Full list of author information is available at the end of the article

The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Sabra et al. BMC Neurology (2016) 16:254
Despite the effectiveness of aspirin in the primary and
secondary prevention of atherothrombotic disease,
patients continue to suffer recurrent thromboembolic
vascular events whilst on AP treatment [16]. This recur-
rence has been associated with high on-treatment plate-
let reactivity (HPR), with up to more than 60% of
subjects being reported to be resistant to antiplatelet
therapy (aspirin or clopidogrel) [17, 18]. Although some
studies showed a link between HPR and major adverse
vascular events [19, 20], the use of platelet function ana-
lysis to detect and manage HPR continues to be debated.
Most studies that investigated HPR were undertaken for
patients already receiving antiplatelet therapy without
assessing baseline platelet reactivity [19, 20], which may
partly explain the conflicting results. We were therefore
interested in whether patients have a higher platelet re-
activity at baseline, which may contribute to the subopti-
mal response to treatment. Hence this study aimed to
compare platelet function, as determined by MEA, in
healthy subjects and stroke patients prior to treatment
initiation and between patients naive to AP therapy and
those on regular low dose aspirin or clopidogrel.
Methods
Study design
A prospective observational study to compare platelet
function between age matched healthy controls and
patients with ischaemic stroke using MEA; and between
patients naive to and on baseline AP therapy.
Patient population
First time ischaemic stroke patients were recruited upon
their presentation to the Emergency Department of a
large teaching hospital (ABMU Health Board, Swansea,
UK). Once a provisional diagnosis of stroke was made
by the care team, strict inclusion criteria were applied.
The inclusion criteria including only adults (18 years),
the clinical assessment of ischaemic stroke was based on
clinical history, examination and neuroradiology, the
diagnosis of stroke was further validated by a member of
the research team using WHO diagnostic criteria [21].
Full informed conset was sought from the outset and for
those unable to consent due to lack of mental capacity
assent was sought from personal or professional legal
representatives. Exclusion criteria included: previous
stroke; reciveing anticoagulant therapy; use of nonsteroidal
anti-inflammatory drugs (NSAIDs) aside from low-dose as-
pirin; suffering from a disease known to alter coagulation
(e.g. liver disease, malignancy, renal failure) or imminent
death. Investigators were blinded to the result of MEA test-
ing. Stroke patients were compared to an age-matched
control group recruited from a healthy local population
who were subsequently tested at the Haemostasis Biomed-
ical Research Unit (HBRU). The helathy volunteers were
Page 2 of 8
recruited via various advertising means including posters,
internal email or direct invitations of staff and patients
relatives. Healthy volunteers were recruited if over 18 years
of age without any significant co-morbidities aside from
mild conditions not likely to affect platelet function (e.g.
mild asthma or dyspepsia) and they were excluded if they
had taken any NSAIDs or other agents that could affect
platelet activity in the 2 weeks prior to testing.
Blood sampling and data collection
One venous blood sample was collected from stroke
patients on admission before any treatment given (base-
line). Blood samples were also collected at 24 h and 35
days after standard treatment (aspirin 300 mg daily, or
thrombolysis followed by aspirin 300 mg after the 24-h
imaging to rule out intracerebral bleeding). The first
2 mL blood was discarded. Blood was then collected in
3 mL tubes containing 25 g/mL hirudin (Dynabyte,
Munich, Germany) as per the manufacturers recom-
mendations. Further blood samples were taken for Full
Blood Count (FBC), Prothrombin Time (PT), activated
partial thromboplastin time (APTT) and fibrinogen
levels. FBC samples were analysed on a Sysmex XE 2100
(TOA Medical Electronics) automated haematology ana-
lyser and routine clotting testing was undertaken using
Sysmex CA1500 analyser. Fibrinogen concentrations
were measured by clauss method. The analysers were
calibrated according to manufacturers instructions and
fibrinogen calibration was checked against the second
International Fibrinogen Standard Version 4 (NIBSC
code 96/612). All reagents were obtained from Dade
Behring.
Multiple electrode aggregometry
Multiple electrode aggregometry (MEA) is a method that
tests platelet function in whole blood, based on whole
blood impedance aggregometry. In this study the Multi-
plate analyser (Dynabyte GmBH, Munich, Germany) was
used. The Multiplate has five testing arease which can be
loaded with the MEA test cells, each of the test cells has
two independent sensor units which are made of two
silver-coated, highly conductive copper wires. The Multi-
plate works by measuring platelet adhesion and aggrega-
tion to these conductive wires following activation of the
platelets. As aggragtaion increases there is an increase in
electrical impedance between the wires which is recorded
on the Multiplate device [22]. Platelet aggregation deter-
mined by MEA is calculated from the Area under the
Curve (AUC) which is taken from the measured electrical
impedance and quantified by arbitrary aggregation units
over time (AU*min) (Fig. 1).
Hirudinated whole blood was kept at room temperature
for 30 min before testing platelet aggregation using the
Multiplate analyser. A volume of 300 l hirudinated
Sabra et al. BMC Neurology (2016) 16:254
Fig. 1 A graph of ADP (channel 1), ASPI (channel 2) and Collagen (channel 3) agonist testing in a patient with stroke. The aggregation is the
increase of impedance during analysis. The velocity is the maximum slope of the aggregation curve. These 2 parameters determine the area
under the curve from what the arbitrary aggregation units over time (AU*min) are calculated with values of between 0 and 200. The left side bar
shows if values are within the manufacturers normal ranges. For this patient only collagen is within normal range but ADP and ASPI values
are raised
blood was added to 300 l of saline at 37 C and allowed
to incubate for 3 min in individual test cells. Then the
following specific agonists were added to respective test
cells and electrical impedance measured:
 Adenosine diphosphate (ADP, 20 l of 0.2 mM stock
solution) which triggers platelet activation via
platelet ADP receptors (i.e. P2Y12 receptor that is
inhibited by clopidogrel).
 ASPI test reagent (20 l of 15 mM stock solution)
contains arachidonic acid. This triggers platelet
aggregation via platelet cyclooxygenase, which is
blocked by aspirin.
 Collagen (20 l of 100 g/ml stock solution) which
activates platelets via the collagen receptor.
HPR has been defined based on the manufacturers
recommendations and published data with cut-offs of
50 AU*min for ADP-induced [19, 23] and 40 AU*min
for ASPI-induced aggregation [24].
Statistical analysis
To establish the baseline characteristics of the healthy
and stroke groups descriptive analyses were performed.
All the data was assessed for normality using normal
probability plots and ShapiroWilk test of normality.
Percentages are used to summerize categorical variables
and chi-square tests were used to assess any difference
between the groups. Continuous variables are presented
as mean and standard deviation (SD) unless otherwise
stated. Differences were assumed to be significant at 5%
level. Two-sample Students t-test were used to compare
differences between different groups. Any differences
were then evaluated by using analysis of covariance
(ANCOVA) whilst controlling for the effect of smoking.
IBM SPSS Statistics for Windows, Version 22.0 (IBM
Page 3 of 8
Corp. Released 2013, Armonk, NY) was used to perform
the analysis.
Results
One hundred twenty two patients with suspected first-ever
ischaemic stroke were recruited between May 2012 and
February 2014. 52 patients were excluded. The reason for
exclusion included: 18 had stroke mimics such as Bells
palsy; sepsis and brain tumour; 8 transient ischemic attacks
(TIA); 8 had a haemorrhagic stroke; 4 previous strokes; 3
cancers; 1 renal failure; 6 found to have been given loading
aspirin prior to blood collection and 4 were taking
NSAIDs such as Ibuprofen and Diclofenac. Leaving
seventy patients (42 men and 28 women; mean age,
73.1 13.3 years) with confirmed first time ischaemic
stroke, these patients were used in the analysis. Their
baseline characteristics are presented in Table 1.
Stroke patients were matched for sex and age with 72
healthy subjects (mean age, 71.3 6.9 years, p = 0.29;
40 men and 32 women, 0.59) recruited from the same
population as the stroke patients. Whilst matched for
age and sex the stroke group did have a significantly
higher proportion of current smokers than healthy
controls (22.9% versus 6.9%, p = 0.01).
Full blood count and coagulation profile results are pre-
sented in Table 2 for all groups. Of the 70 patients, 31
were thrombolysed and 39 were loaded with 300 mg as-
pirin. At baseline 33 stroke patients were on antiplatelet
therapy (26 on 75 mg of aspirin only, 3 on 75 mg of clopi-
dogrel only and 4 on both agents) and 37 were AP-naive.
Multiplate analysis showed that ASPI was the only
significant parameter that differed between the healthy
subjects and the entire stroke group (97.6 27.3 versus
81.1 50.1, p = 0.02). These differences reversed when
healthy subjects were only compared with AP-naive
patients (97.6 27.3 versus 117.4 30.7, p < 0.005). In
Sabra et al. BMC Neurology (2016) 16:254 Page 4 of 8

Table 1 Baseline characteristics of patients with stroke (n =70) Table 3 Results of MEA for stroke patients and healthy subjects
Age, mean SD 73.1 13.3 Group Healthy (n 72) Stroke (n 70)
Sex: male/female 42/28 All AP-nave (n 37) On AP (n 33)
Current Smoker 16 (22.9%) ADP 81.3 24.3 89.8 30.1 98.2 31.1** 80.3 26.4
Hypertension 48 (68.6%) ASPI 97.6 27.3 81.1 50.1* 117.4 30.7** 40.4 33.2***
Ischaemic heart disease 21 (30%) Collagen 82.4 20.2 82.8 29.1 99.5 25** 64.1 20.9***
Atrial fibrillation 18 (25.7%) Values reported as mean and standard deviation, AP antiplatelet naive at baseline
*p < 0.05 compared with healthy controls
Diabetes mellitus 14 (20%) **p < 0.005 compared with healthy controls
***p < 0.00005 compared with healthy controls
Previous TIA 11 (15.7%) p < 0.05 compared with AP naive group
Hyperlipidaemia 27 (38.6%) p < 0.0000005 compared with AP naive group

Antiplatelets 33 (47.1%)
Aspirin only 26 Prevalence of high on-treatment platelet reactivity (HPR)
Clopidogrel only 3 Based on the cut off values for HPR, 10 patients
Both drugs 4 (33%) on long term aspirin (baseline sample) were
Statins 26 (37.1%) aspirin-resistant or low responders but only 4 had
Glucose (mmol/L) 6.5 1.7 ASPI values > 60 AU*min. Of the seven patients on clopi-
Creatinine (mol/L) 97 27.2 dogrel at baseline, 5 (71%) were clopidogrel-resistant
(4 patients had ADP values above 85 AU*min).
D-dimer (ng/mL) 208 [104488]
ASPI results were available for 36 patients at 35 days
C-reactive protein (mg/L) 4 [110]
after 300 mg loading doses of aspirin. 13.6% (3/22) as-
TOAST classification: pirin naive patients and 7.1% (1/14) on long term aspirin
Large artery 20 were considered aspirin-resistant but only one patient
Cardio-embolic 14 (1/36) had ASPI values > 60 AU*min. At 24 h, it is
Small vessel (lacunar) 29
Undetermined aetiology 7 understandable that for thrombolysis patients (dotted
Values are presented as percentages, mean SD or median
lines, Fig. 2) ASPI values were higher than aspirin group
(interquartile range) (solid lines) because they have not received any aspirin
pending the 24 h head CT scan to rule out intracerebral
haemorrhage. The results of serial ASPI testing at
the AP-naive patients, significantly higher values were baseline, 24 h and 35 days for patients who were
also observed for ADP and Collagen as shown in aspirin-naive or on long term aspirin are shown in
Table 3. All MEA parameters were significantly lower Fig. 2.
in patients who were on regular antiplatelet therapy
at baseline as compared to healthy subjects and AP- Discussion
naive patients (Table 3). All these differences contin- Our study is the first to assess platelet reactivity using
ued to be significant after controlling for smoking MEA in patients with stroke at baseline before loading
status using ANCOVA. doses of 300 mg aspirin were given. The main finding
showed that platelet aggregation in AP-naive patients is
higher than in healthy subjects for all pathways investi-
gated. However, it is difficult to ascertain if this is due to
Table 2 Haematological tests for stroke patients and healthy an underlying high platelet reactivity or is secondary to
subjects the acute event. Future larger studies are needed to
Group Healthy (n 72) Stroke (n 70) confirm our findings and investigate this assumption.
All AP- naive On AP Secondly, our study detected changes between long-
Plt (x109/l) 245 50 256 79 271.3 83 238 71 term aspirin users and aspirin-naive patients. Despite
Hb (g/dl) 14.3 1.3 14.2 1.7 14.4 1.6 13.9 2 the proved effectiveness of AP therapy, patients continue
HCT (g/l) 0.43 0.04 0.42 0.04 0.42 0.04 0.41 0.05 to have recurrent vascular events while on treatment
[16]. In agreement with previously reported studies [25],
PT (secs) 10.6 0.7 10.6 0.6 10.5 0.5 10.8 0.7
about 50% of patients in our study were taking one or
APTT (secs) 26 2.3 24.2 2.2* 23.9 2* 24.4 2.3*
more AP drugs at presentation but this did not prevent
FBG (g/l) 3.2 0.5 3.8 0.8* 3.9 0.9* 3.7 0.7* stroke. New approaches to improve clinical outcomes
Values reported as mean and standard deviation, AP antiplatelet naive used MEA to individualise antiplatelet therapy [19] and
at baseline
*p < 0.05 compared with healthy controls
hence highlighting the importance of our findings. Based
p < 0.05 compared with AP naive group on definition of aspirin resistance in previous studies,
Sabra et al. BMC Neurology (2016) 16:254 Page 5 of 8

Fig. 2 Distribution of ASPI-induced aggregation at baseline, 24 h and 35 days after 300 mg loading doses with aspirin. a In aspirin-naive patients,
13.6% had HPR (non-responder) at 35days. b Of patients on long term aspirin, only 7.1% were non responders at 35days. Horizontal dotted
black lines indicate ASPI cut-off values for aspirin resistance, as reported in the literature

33.3% of patients would be defined as aspirin-resistant
according to ASPI results. As more than 60% of long
term aspirin users are considered responders, the use of
loading dose of aspirin in these patients may not have
any additional benefit. It may indicate the need to inves-
tigate thoroughly for underlying silent risk factors such
as paroxysmal atrial fibrillation. The emergence of new
P2Y12 antagonists and ongoing uncertainties of platelet
function testing dictate the need for larger RCT to an-
swer these questions and investigate the clinical benefit
of AP switching strategy in stroke patients based on
MEA results. This supports the recommendation by the
National Institute for Health and Care Excellence for fu-
ture research in this area about effectiveness and safety
of clopidogrel, alone or in combination with aspirin, in
treating stroke and TIA [26].
Thirdly, our study also detected the effect of aspirin
loading therapy on platelet aggregation (ASPI) in acute
stroke. Interestingly at 35 days after in hospital ad-
ministration of aspirin, only 11.1% were non or low
responders. Therefore, there is a discrepancy of more
than 20% in presumed aspirin-resistance rates between
self-reported and in hospital aspirin intake. Many factors
may explain this discrepancy including poor compliance,
timing of testing following aspirin intake and low-dose
versus high-dose aspirin. Up to 50% of patients are
reported to be poorly compliant [27, 28]. Although many
strategies are in place to encourage the public to
Sabra et al. BMC Neurology (2016) 16:254
recognise and act on stroke symptoms, little has been
done to improve compliance. Previous studies suggested
female sex, single marital status, lower education level,
depression, diabetes mellitus, polypharmacy, and smok-
ing as predictors of poor compliance [27]. Hence, more
educational initiatives targeting these high-risk patients
of poor compliance should be done to enforce the
importance of taking AP regularly. Another possible
explanation of this discrepancy is the timing of baseline
sample after aspirin intake, which was variable, whereas
it was 24 h after administering aspirin for subsequent
samples. Timing of platelet function testing has been
acknowledged as a factor explaining the variability in the
results with serum salicylate level peaking at 1 h after
administration [5, 29]. The higher aspirin dose given
acutely in hospital is another potential factor but pub-
lished data do not support this assumption reporting
that doses between 30 and 1300 mg per day have the
same clinical efficacy [18, 30]. Nonetheless some may
argue that this does not equate to similar platelet inhib-
ition as detected by platelet function tests.
Despite the availability of a wide-range of techniques
to investigate platelet function, their reliability has been
questioned. A previous study showed poor correlation
between six platelet function tests to define aspirin-
resistance [31], and this variation in methodology may
be a factor in explaining the difficulties in determining
the true prevalence of AP resistance. Hence it is import-
ant to link these tests to clinical endpoints to establish
cut-offs of each assay. Although LTA may be considered
the gold standard for the determination of platelet activ-
ity, we chose to use Multiplate in this study because it
is a standardized fully automated point-of-care test that
is relatively easy to use without many of the technical
limitations of the previous assays [32]. Additionally the
feasibility of using Multiplate in the context of routine
hospital service has been ascertained to provide repro-
ducible and precise results in stroke patients [33]. This
potentially makes our findings easier to replicate and
translate into large clinical studies to investigate clinical
endpoints.
Limitations: this is a small single centre study and
hence may not be generalizable to other populations.
Aspirin and clopidogrel intake at baseline was self-
reported by patients or their family. Although MEA
results indicate two thirds of the patients were taking
aspirin, confirming compliance is an important issue but
was beyond the scope of this study. Another limitation
is the higher number of active smokers in the stroke
group as compared to healthy subjects. Conflicting
results exist about the effect of smoking on platelet
function testing [20, 24, 34]. However in our study,
ANCOVA analysis was used to control for the effect of
smoking. Another limitation is the attrition rate despite
Page 6 of 8
the prospective study design. Multiplate analysis at 35
days was not possible for all cases for various reasons
(patient discharged or transferred to another hospital,
death, declined further samples, difficult to bleed).
Despite the strict inclusion and exclusion criteria hetero-
geneity of stroke presentations and patient populations
are key issues to consider when investigating stroke as
noticed in this study, and subgroup analysis should be
interpreted with caution.
Conclusions
Despite these limitations our study reports a higher
platelet reactivity as determined by Multiplate in AP-
naive stroke patients for all pathways as compared to
healthy subjects. Many patients receiving low dose
aspirin met the criteria of HPR or drug resistance but
the prevalence was much lower at 35 days of inpatient
treatment. It also reiterates the importance of compli-
ance and probably timing of testing as potential factors
affecting MEA testing. It also questions the benefits of
giving aspirin in patients with laboratory evidence of
response when this did not prevent recurrent stroke.
Hence further work in large prospective multicentre
trials is needed to confirm that the laboratory results
can be translated onto clinical endpoints. For any future
research the main question is whether the use of MEA
to individualise AP treatment in stroke/TIA results in
better clinical outcomes. We recommend establishing
platelet reactivity at baseline to determine best AP therapy
and then monitor their effect at 3 days to decide whether
a switch is warranted. This may also help establish the
true causes of HPR in stroke and the value of MEA in
predicting future events.
Abbreviations
ADP: Adenosine diphosphate; ANCOVA: Analysis of covariance;
AP: Antiplatelet drugs; ASPI: Arachidonic acid; AU*min: Aggregation units over
time; AUC: Area under the curve; COL: Collagen; HPR: High on-treatment plate-
let reactivity; LTA: Light transmission aggregometry; MEA: Multiple electrode
platelet aggregometry; NSAIDs: Nonsteroidal anti-inflammatory drugs;
RCT: Randomised control trial; SD: Standard deviation; TIA: Transient ischaemic
attack
Acknowledgments
Our thanks go to the staff in the Emergency and Stroke Department and the
research team in NISCHR Haemostasis Biomedical Research Unit at Moriston
Hospital for their support.
Funding
This study was funded by the National Institute for Social Care and Clinical
Health Research (NISCHR).
Availability of data and materials
The datasets generated and/or analyzed during this study are not publicly
available because they contain protected health information but de-identified
data are available from the corresponding author on reasonable request.
Sabra et al. BMC Neurology (2016) 16:254
Authors contributions
PAE conceived the study. AS, SNS, ML and PAE designed and coordinated
the study. AS, SNS and PAE drafted the article and interpreted the data. AS,
SNS, SS and LD recruited the patients and collected blood sample. AS, SNS,
ML and LD performed the rheology tests. ML provided rheological advice.
SNS, SS and VE collected the patient data. AS and RHKM performed the
statistical analyses and interpreted the data. MW and JFP revised the
manuscript critically for important intellectual content. All authors read and
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
The study has been approved by the local ethics committee (South West
Wales Research Ethics Committee). Written informed consent has been
obtained for all patients.
Author details
1
Medical School, Swansea University, Swansea, UK. 2NISCHR Haemostasis
Biomedical Research Unit, Morriston Hospital, Swansea, UK. 3Cardiology
Department, Singleton Hospital, ABMU Health Board, Swansea, UK. 4School
of Applied Sciences, Cardiff Metropolitan University, Cardiff, UK. 5Department
of Stroke Medicine, Morriston Hospital, Swansea, UK. 6Norwich Medical
School, University of East Anglia, Norwich, UK. 7Emergency Department,
Morriston Hospital, ABMU Health Board, Swansea SA6 6NL, UK.
Received: 15 August 2016 Accepted: 5 December 2016
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